The objectives of this project are to understand the mechanism of the Na-K pump through the study of the structure of the Na-K ATPase, the conformational dynamics of its active sites and the perturbations cased by cardiac glycosides with fluorescence spectroscopy. Available compounds, such as the anthroyl-ouabain I synthesized and derivatives of ATP, will be used as site-specific fluorescent probes for steady state and nanosecond fluorescence spectroscopy studies. Syntheses of new specific fluorescent studies will provide information on the molecular properties of the labeled sites, their changes in different functional states, the perturbations caused by inhibitors and their location with respect to the membrane and each other. Energy transfer between appropriate pairs of chromophores bound at specific sites on the enzyme will be studied attempting to measure the distance between the active sites, their changes in conformation and the possible subunit structure of the pump complex. The possibility of studying the Na-K ATPase with fluorescence microscopy of living cells in culture and of measuring diffusion of the enzyme in the plane of the membrane will be investigated. The results of these studies may increase our understanding of the structure and mechanisms of the Na-K pump and the mode of action of cardiac glycosides. In addition it is expected that new spectroscopic methods that will allow continuous monitoring of Na-K ATPase conformation, kinetics and equilibrium of ligand binding will be developed. The approaches, methods and findings in this work may have general applicability in the study of other membrane proteins and in screening normal and pathologic function in living cells.